Retrospective Study On The Correlation Between DNA Repair System And Its Polymorphism With Breast Cancer

Part I. The correlation between DNA repair system and breast cancerBackground and Objective Breast cancer is one of the most common tumors in women at present of the world, and it is the major cause of cancer death in women as well. According to the statistics of WHO International Cancer Research Center, in 2008 the new global incidence of female breast cancer cases reached 1380000, accounting for all female malignant tumor incidence of 22.9%; 460000 women dying from breast cancer, accounting for the death of all female malignant tumors accounted for 13.7%, and the death of 1.7%. In 2010, there were 207090 new breast cancer cases and 39840 deaths from the disease in the USA. Heterogeneity in breast cancer diagnosis, treatment and survival may be partially explained by breast biology and malignant phenotype. Utilization of mammography screening has dramatically decreased the rates of advanced breast cancer, particularly in postmenopausal women and increased years of life after surgery or other treatment. In contrast, premenopausal women have not benefited from early detection to the extent of age 50 women. Increasing evidence suggests etiological and mechanistic differences in breast cancer development in young women as well as in African-Americans. Based on the statistics, the annual female breast cancer in China is 169000, being the second most common malignant tumor of women; and female breast cancer death in China is about 45000, the sixth death cause of malignant tumor in women. With the treatment methods of screening and diagnosis of breast imaging technology, comprehensive application makes the mortality rate of breast cancer decreased gradually, but brings the problem of loss of patient economic burden and quality of life. Breast cancer pathogenesis has not been fully elucidated, but current researches suggest the changes and development of various kinds of environmental factors, social factors, human lifestyle changes as well as some genes are closely related to the occurrence of breast cancer. If the induced breast cancer risk factors can be recognized and controlled, it will play a positive role in breast cancer prevention.Over the past three decades numerous studies have investigated the association of lifestyle with breast cancer, showing independent effects of various factors. A summary of the present state of knowledge on the role of lifestyle patterns have been identified, such as physical activity, diet, smoking, hormone therapy, and experience of psychological stress in the modulation of breast cancer in women, and discuss commonly accepted biological mechanisms hypothesized as responsible for the associations. The findings indicate that regular physical activity of moderate to vigorous intensity is probably linked with the decreased breast cancer risk among postmenopausal females and suggestive for a decrease of the risk in premenopausal women. In contrast, the consumption of high-fat diet, alcohol intake, and use of combined estrogen and synthetic progestagen hormonal therapy may increase the risk. Epidemiological findings have been proved the mentions above. The cancer is a multistep process and several risk factors have been recognized. Several human genetic and heredity factors have the relations with breast cancer.Every day, about 105 cells of human beings are experiencing a variety of lesions; these lesions are composed of natural decline, replication error, cell metabolism and exposure to radiation or chemicals. In order to maintain the survival and function of cells, lesion cells must be repaired by the specific repair mechanism. DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. DNA damage is induced by exposure to environmental agents and is generated spontaneously during normal cellular metabolism. Reactive oxygen species (ROS) are an unavoidable by-product of aerobic metabolism and cause both base damage and strand breaks. Additional spontaneous cellular reactions include the hydrolytic loss of bases, especially purines, from the phosphodiester backbone, as well as the deamination and alkylation of bases. In humans, it has been estimated that up to 100,000 spontaneous DNA lesions are generated daily per cell. Environmental DNA-damaging agents include the ultraviolet (UV) component of sunlight, which generates cyclobutane pyrimidine dimers and oxidative base damage; ionizing radiation, which produces clusters of ROS that create double-strand DNA breaks; and base-damaging chemicals such as aflatoxins, benzo (a) pyrene, methyl chloride, and nitrosamines, which alter or destroy base-pairing capacity. Because DNA damage has the potential to inhibit and/or alter fidelity of replication and transcription, there is a need for diverse and highly accurate repair processes. There is also a need for bypass mechanisms that allow unrepaired damage to be tolerated if encountered during replication. An emerging theme in the past 20 years is that there is considerable overlap between the various repair and bypass pathways in terms of the cognate lesions that each can deal with. This functional redundancy is partially a reflection of the very high load of endogenous DNA damage and underscores the importance of these pathways in the maintenance of genome stability.DNA damage repair mechanism consists of nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and double strand break repair (DSBR). The NER pathway repair the chemicals and ionizing radiation induced double helix distorting lesions. NER is characterized by an ability to remove a large number of structurally unrelated, helix-distorting lesions that interfere with base pairing and generally impair replication and transcription. This pathway is particularly relevant for preventing the lethal and mutagenic effects of environmental mutagens; cognate lesions include the UV-induced CPD and 6-4 photoproduct as well as chemical carcinogen-induced bulky adducts. Loss of NER in humans is associated with the disease xeroderma pigmentosum, which is characterized by an extreme sensitivity to sunlight and cancer predisposition. NER also removes bulky, endogenous oxidative DNA damage that results from intramolecular crosslinking between the C8 position of purines and the 59 position of deoxyribose. Finally, NER can provide an alternative mechanism to repair AP sites and oxidized bases. BER pathway repair for small chemical injury. The major endogenous DNA damages result from oxidative stress, hydrolysis, or deamination and are removed by the base excision repair pathway. BER requires the sequential action of five DNA-modifying activities, a DNA N-glycosylase that releases the base from deoxyribose, and finlly DNA ligase to seal the remaining nick. MMR pathway repair to fix the errors during the copy process. The mismatch repair (MMR) pathway removes helical distortions that arise when errors are made during DNA synthesis or when non-identical duplexes exchange strands during recombination. During replication, mismatch correction limits mutagenesis; during recombination, correction of mismatches within heteroduplex DNA intermediates generates gene conversion events. In addition to initiating mismatch removal during recombination, the MMR system monitors identity between interacting molecules, which can limit use of non-identical substrates as repair templates. The DSBR pathway is mainly aimed at the different stages of the cell cycle, the repair pathway involving DNA damage induced by chemotherapy and radiotherapy. Repair of DNA DSBR is mediated by the HR and NHEJ pathways. HR involves the Rad52 group of proteins, BRCA1/2 and XRCC2/3, in addition to EME1 and NBS1. Multiple hereditary disorders have been associated with defects in HR, including mutations in BRCA1 or BRCA2, which have been associated with hereditary breast and ovarian cancer. In contrast, DSBR repair via the NHEJ pathway is potentially more mutagenic and requires the coordinated assembly of a number of proteins at the DNA termini to facilitate end joining. These proteins include the Ku hetero-dimer (Ku70/Ku80), DNA protein kinase catalytic subunit (DNA-PKcs), ligase IV, XRCC4, XLF, Artemis, and polymerases and 1. Mutations in NHEJ components have been described and confer radiation sensitivity and defective immune function via reduced V(D)J recombination, whereas complete abrogation of NHEJ components seems to be incompatible with life. The role of NHEJ and HR in the repair of ionizing radiation-induced DNA damage has also made these pathways popular as targets for the development of radio-sensitizing agents. DNA DSBR can also arise via the enzymatic processing of interstrand DNA cross-links. DSBR pathway for repair DNA replication in cells begin to enter S phase. DSBR can repair of reactive oxygen species, ionizing radiation or chemical substance such as topoisomerase Ⅰ inhibitor or double stranded DNA intermediates. For the NHEJ and HR pathways selection depend mainly on the complex regulation mechanism and relates to the 53BP1 and BRCA1 genes.The present results indicate that, the occurrence of DNA damage repair mechanism and breast cancer has close correlation. Risk of the high level deletion of DNA damage and DNA repair mechanism is related to the breast cancer. The comparative studies have shown the cases of breast cancer in young women, a decline in DNA repair capacity increased the risk of illness. Therefore, it is very important to study the relations of the breast caner with DNA repair pathways, and the polymorism analysis of the DNA repair pathways genes with breast cancer for theory and clinical treatment significance.Prior to the DNA repair gene research in breast cancer have been reported, but there is no systematic elaboration analysis of the correlation between these four types of repair genes with breast cancer. Based on this point, the comprehensive study for correlations between four DNA repair gene systems with breast cancer relationships are performed.The correlation of four types of DNA repair mechanisms and breast cancer, including NER for ERCC2 (excision repair cross-complementation group 2), ERCC4 (excision repair cross-complementation group 4), ERCC5 (excision repair cross-complementation group 5) and XPC (xeroderma pigmentosum group C); the BER for ADPRT (poly (ADP-ribose) polymerase-1), APE1 (Apurinic/apyrimidinic endonuclease 1), XRCC1 (X-ray cross complementing group 1) and POLD1 (DNA polymerase 8); the MMR for MLH1 (the human MutL homologue), MSH3 (MutS homolog 3) and MSH6 (MutS homolog 6) and DSBR for NBS1 (Nijmegen breakage syndrome 1) and XRCC3 (X-ray cross complementing group3).Materials and Methods The hospital treated a total of 360 cases of breast cancer patients in the study as the research object, makes a comparison between mRNA and protein expression through the acquisition process in the treatment of breast cancer tissue samples and 360 cases normal tissue from others in the same hospital, correlation analysis of four types of DNA repair genes and breast cancer.Results 1.NER pathway, the mRNA and protein expression of ERCC2, ERCC4 and ERCC5 in breast cancer and normal breast tissue level have no significant difference (P>0.05); XPC gene mRNA and protein expression levels with normal breast tissues had statistical difference (t-XPC-mRNA=60.884, P XPC-mRNA=0.000; χ2 XPC-protein=39.057, PXPC-protein=0.000), compared with normal tissues of the mRNA and protein levels were decreased.2. BER pathway, the mRNA and protein expression of ADPRT, APE1 and POLD1 in breast cancer and normal breast tissue level comparisons showed statistical difference (t-ADPRT-mRNA=91.275, PADPRT-mRNA=0.000; t-APE1-mRNA=113.151, PAPE1-mRNA=0.000; t-POLD1-mRNA=102.587, PPOLD1-mRNA=0.000;χ2ADPRT-protein=26.639, PADPRT-protein=0.000; χ2APE1-protein=29.743, PAPE1-protein=0.0001;χ2POLDl-protein=42.279, PPOLD1-protein=0.000), the expression of mRNA and protein in cancer tissue increased than normal tissue; the expression of mRNA and protein of XRCC1 gene compared with normal breast tissue levels also had statistical difference (t-xRCC1-mRNA= 164.251, PXRCC1-mRNA=0.000; χ2xRCC1-protein=59.854, PXRCC1-protein=0.000), however, compared with the normal tissue the mRNA increased while protein level decreased.3. MMR pathway, the mRNA and protein expression of MSH3 and MSH6 in breast cancer and normal breast tissue level have no significant difference (P>0.05); the expression of mRNA and protein of MLH1 gene comparison of the levels and normal breast tissue had statistical difference (t-MLH1-mRNA-100.84, PMLH1-mRNA=0.000; χ2MLH1-protein=44.066, PMLH1-protein=0.000), compared with the normal tissue levels of mRNA increased and protein expression level decreased.4. DSBR pathway, the mRNA and protein expression of NBS1 in the in breast cancer tissue and expression of normal breast tissue were statistically significant difference (t-NBS1-mRNA=143.211, PNBS1-mRNA=0.000; χ2NBSi-protein=38.172, PMLH1-protein=0.000), the mRNA and protein expression in cancer tissue the elevated than normal tissue; mRNA and protein expression of XRCC3 gene level showed no significant difference compared with normal breast tissue (P>0.05).Conclusions The expression of mRNA and protein of gene for DNA repair pathways in breast cancer have obvious differences, DNA repair pathways of the same type in the cancer tissues is not consistent as well.Part Ⅱ. The polymorphism analysis for DNA repair system genes with breast cancerBackground and Objective Breast cancer is one of the most common tumors in today’s world of women, and it is the major cause of cancer death in women. The global annual increase of cases of breast cancer is about 1000000 new cases are mainly from Europe and the United States, including 200000 in the United States, accounting for malignant tumors in female 27%; Europe 320000, accounting for 31% of female malignant tumor. From twentieth Century since 70, originally as a low incidence area of breast cancer incidence rates in Asia have been on the rise, especially in Japan, Singapore and China’s coastal cities. Breast cancer is a complex disease; in addition to familial breast cancer is mainly concerned with BRCA1 and BRCA2, and may be environmental factors in sporadic breast cancer related. The genetic susceptibility to cancer at the molecular level for the occurrence and development of breast cancer plays an important role in.Differences between individual gene nucleotide sequences in the crowd called gene polymorphism. Gene polymorphisms include DNA polymorphisms and length polymorphism. Differences in allele specific sites on DNA sequence is called polymorphism, including point mutation, transversions and transitions, a single base deletion, substitution and insertion. The frequency distribution of the normal individual genomic DNA crowd single nucleotide sequence differences in the more than 1% known as the SNP single nucleotide polymorphism. SNP is the study of human genome project in a new genetic marker. The cause of gene polymorphism is mainly alleles and co dominance etc. SNP single nucleotide is the abbreviation of the polymorphism, is refers in the genome of single nucleotide mutation, genetic marker formation, its quantity many, abundant polymorphism. Genome single nucleotide variation, include replacement, transversion, insertion and deletion. SNP is very widely distributed in the genome; recent studies indicate that in the human genome per 300 base pairs will appear once. The existence of a large number of SNP sites, so that people have the opportunity to discover and a variety of diseases, including cancer genome related mutations; from the experimental operation point of view, through the SNP found that the disease associated gene mutation to be easier than the family; some SNP does not directly lead to the expression of disease genes, but because of its gene and some diseases of adjacent, and become an important mark. SNP in basic research has played a great role.The present study shows that had close relation with occurrence, development of nucleotide polymorphism of several important genes and breast cancer. For example: cytokine gene polymorphism in breast cancer related is considered to be the potential risk factors, cytokines and tumor is a hot cancer molecular epidemiology study. Cytokine because of its broad effect, both plays an important role in the pathogenesis and prognosis of breast cancer. Another aspect is the cytochrome P450. Cytochrome P450 (CYP450) is a kind of gene superfamily enzyme system, its function is to catalyze oxidation reaction stage of foreign compounds enter the body. For chemicalcarcinogens, which no active pre carcinogens activated and transformed to electrophilic compounds, electrophilic compounds of biological macromolecules attack inside the cell, formed with DNA or protein adducts, ultimately causing oncogene and tumor suppressor gene change, leading to cancer. And the third aspects of the relationship between the polymorphisms of DNA repair genes involved in the system and the susceptibility of breast cancer, the results of the present study have shown that XRCC1, with a statistical association between polymorphism of Rad51 and NBS1 genes and breast cancer susceptibility.Danger of high levels of DNA damage and DNA repair deficiency and breast cancer is highly related to the. Exposed to different complex DNA damage, integrity of the body needs many repair pathways to maintain genome, which include base excision repair (BER) and nucleotide excision repair (NER), mismatch repair (MMR) and double strand break repair (DSBR). DNA damage repair is one of the important part of defense mechanisms in mammalian cells can be reversed by the body, caused by mutations in the internal and external environmental factors DNA, ensure the stability of genetic material to a great extent. According to the different injury types, DNA repair mechanisms involved in base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA double strand break (DSBR) homologous recombination in (HR) and non homologous end joining (NHEJ) etc. At present, the research on breast cancer susceptibility gene polymorphism results mainly concentrated in a few genes BRCA1, BRCA2, XRCC1, NBS1 and TP53.At present, research on DNA repair gene polymorphism and susceptibility of breast cancer mainly concentrated in the ERCC2, ADPRT and XRCC1 of the 3 gene types, and has obtained the certain research results. But for the relationship between the polymorphism of susceptibility to other types of DNA repair system gene and breast cancer has not been reported. DNA damage repair pathway different plays an important role in maintaining genome stability, and SNPs in a variety of repair pathway mutations in early age and late age of onset and breast cancer are associated with. According to the SNP change can lead to changes in the amino acid substitution and function theory, we speculate that different repair pathways between SNP and breast cancer has the additional or the role and impact of multiplicity. Therefore, the content of this part is to study the correlation analysis between DNA repair pathway gene polymorphism and susceptibility to breast cancer development.Materials and Methods This study evaluated the 18 SNPs of DNA repair system genes with breast cancer as follows:(1) BER:ADPRT V762A (rs1136410), APE1 D148E (rs3136820), XRCC1 R194W(rsl799782)/R280H (rs25489)/R399Q (rs25487)and POLD1 R119H(rs1726801); (2) DSBR:NBS1 E185Q (rs1805794) and XRCC3 T241M(rs861539); (3) MMR:MLH1 I219V (rs1799977), MSH3 R940Q (rs184967)/T1036A (rs26279) and MSH6 G39E (rs1042821) (4) NER: ERCC2 D312N (rs1799793)/K751Q (rs13181), ERCC4 R415Q(rs 1800067), ERCC5 D1104H (rs17655) and XPC A499V(rs2228000)/K939Q (rs2228001). The hospital treated a total of 360 cases of breast cancer patients in the study as the research object, simultaneous acquisition of all patients peripheral venous blood samples was studied and compared for all kinds of repair gene polymorphism, using Sequenom Mass ARRAY time of flight mass spectrometry.Results1.By Hardy-Weinberg equilibrium, the ADPRT V762A, POLD1 R119H、MLH1 I219V、MSH3 R940Q and XRCC3 T241M were not in line with HWE.2. For the single factor analysis of the SNPs of DNA repair genes.(1) NER pathway, the ERCC2 312 NN and DN/NN genotype are risk factors on the incidence of breast cancer (OR=3.66 95%; CI=2.06,6.53 and OR=1.57 95%; CI=1.16,2.10).(2) BER pathway, the XRCC1280 HH genotype is a risk factor on the incidence of breast cancer (OR=2.13 95%; CI=1.34,3.41).(3) DSBR pathway, the NBS1 185 QQ genotype is a risk factor on the incidence of breast cancer (OR=4.33 95%; CI=2.04,9.23).(4) Other DNA repair system genes have no statistical significance with the control (P>0.05).3. For the adjusted results from ages and family history, we perfomed the same analysis for the SNPs of DNA repair genes.(1) NER pathway, the ERCC2 312 DN, NN and DN/NN genotype are risk factors on the incidence of breast cancer (OR=1.62 95%; CI=1.11,2.36; OR=4.90 95%; CI=2.50,9.63 and OR=1.90 95%; CI=1.35,2.69).(2) BER pathway, the XRCC1 280 HH and RH/HH genotype are a risk factors on the incidence of breast cancer (OR=2.50 95%; CI=1.42,4.40 and OR=1.63 95%; CI=1.15,2.30).(3) DSBR pathway, the NBS1 185 QQ and EQ/QQ genotype are risk factors on the incidence of breast cancer (OR=3.87 95%; CI=1.66,9.06 and OR=1.63 95%; CI=1.12,2.35).(4) Other DNA repair system genes have no statistical significance with the control (P>0.05).Conclusions ERCC2, XRCC1 and NBS1 of the 3 genotypes have significance with polymorphisms with breast cancer; the other genotype polymorphism has been found the relationship, which is important significance for the primary prevention of breast cancer.